PROJECT SUMMARY The focus of this proposal is to develop pressure-sensitive T cells that can activate in response to the high interstitial fluid pressure (IFP) in many solid tumors and release an engineered enzyme to neutralize its source. The dense fibrous extracellular matrix (ECM) tissue growth common in many solid tumors presents a physical barrier to the current standard of care chemotherapies and immunotherapies. It leads to the compressive stresses that cause high interstitial fluid pressure (IFP) (>100 mmHg) as compared to that within normal tissues (-4 to -6 mmHg). Together, the ECM barrier and IFP resist the influx of therapeutics into tumor sites. The ECM also harbors the tumor microenvironment (TME) that causes drug resistance and immunosuppression. Hence, the challenge is to selectively break down the ECM at the tumor site to enable the entry of therapeutic cytolytic T cells without affecting the normal connective tissues. Here, the investigators propose that T cells can be engineered to provide a solution to this challenge by selectively degrading the ECM. The investigators' long-term goal is to develop new treatments by harnessing the cell's potential to interact with the in vivo environment and target the underlying mechanisms in the disease pathology. The objective of this project, toward this long-term goal, is to engineer the T cells to synthesize and release the ECM degrading enzymes upon sensing increased IFP. To achieve this objective, their strategy is to engineer the T cells with an artificial cell signaling cascade that upregulates the desired proteins in situ. This work is supported by their preliminary work on engineering the T cells and use of a new assay to simulate IFP in vitro. The rationale for this effort is that it will lead to a cellular technology to locally disrupt the tumor ECM and reduce IFP to assist the influx of antitumor agents for improved treatment outcome. The investigators have described specific milestones with quantitative metric of success and will use the following parallel aims to conduct the investigations: Engineering of T cells with pressure-sensitive trigger (Aim 1); Engineering of T cell for pressure-induced expression of ECM degrading enzyme (Aim 2). This effort is expected to lead to a broad- spectrum technology that has the potential for high-impact. This is because there is no known molecular biomarker to target either the ECM or IFP. The proposed approach is innovative because it challenges the status quo by engineering the T cells to actively traffic to the TME and synthesize an ECM-degrading enzyme in situ. This will mitigate the IFP and enable perfusion of engineered cytolytic T cells to kill tumor cells; as well as alter the continuously evolving TME to overcome drug resistance and immunosuppression.